CRISPR screens identify genes essential for virulence among proteins of hyperLOPIT-unassigned subcellular localization in .

The research field to identify and characterize genes essential for virulence in has been dramatically advanced by a series of clustered regularly interspaced short palindromic repeats (CRISPR) screens. Although subcellular localizations of thousands of proteins were predicted by the spatial proteomic method called hyperLOPIT, those of more than 1,000 proteins remained unassigned, and their essentiality in virulence was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned proteins and performed CRISPR screens. As a result, we identified several genes essential for virulence that were previously unreported. We further characterized two candidates, TgGTPase and TgRimM, which are localized in the cytoplasm and the apicoplast, respectively. Both genes are essential for parasite virulence and widely conserved in the phylum Apicomplexa. Collectively, our current study provides a resource for estimating the essentiality of proteins with previously unknown localizations.IMPORTANCE is a protozoan parasite that causes severe infection in immunocompromised patients or newborns. possesses more than 8,000 genes; however, the genes essential for virulence were not fully identified. The apicomplexan parasites, including , developed unique organelles that do not exist in other model organisms; thus, determining the subcellular location of parasite proteins is important for understanding their functions. Here, we used genetic screens that enabled us to investigate hundreds of genes in during mouse infection. We screened approximately 600 parasite proteins with previously unknown subcellular localizations. We identified many novel genes that confer parasite virulence in mice. Among the top hits, we characterized two genes essential for virulence, TgGTPase and TgRimM, which are widely conserved in the phylum Apicomplexa. Our findings will contribute to understanding how apicomplexans adapt to the host environment and cause disease.